Abstract The long-term goal of this research is to improve the clinical outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) by improving red blood cell (RBC) transfusion practices in stem cell transplant recipients. Recent studies from our PPG collaborators indicate significant metabolmic changes in RBC units during storage that may lead to adverse effects in transfusion recipients. A better understanding of how RBC transfusions influence the activation, proliferation, and graft-versus-host disease (GvHD) activity of donor T- cells is needed, as this represents a significant gap in our current understanding of the immunology of allogeneic transplantation. To address this gap, we developed novel P ?? F1 models of murine allo-HSCT and RBC transfusion, using defined conditions of red cell collection and storage. Preliminary data demonstrate a modulatory effect of red cell transfusions on the activation and proliferation of donor T-cells in transplant recipients: fresh RBC transfusions increased the observed incidence of GvHD while transfusion of very old stored RBC supressed the activation of donor T-cells and markedly reduce GvHD moratilty in murine allo- HSCT recipients. A retrospective analysis of 345 allo-HSCT patients demonstrated a significantly increased risk of grade 3-4 acute GvHD in patients who received more RBC transfusions using a multivariable model that controlled for other clinical factors associated with GvHD, and censored transfusions performed after the diagnosis of GvHD. This proposal builds upon complementary preliminary data from murine and human studies to test the overall hypothesis that early post-transplant transfusion of allogeneic red cells to HSCT recipients leads to the activation of donor T-cells and increased GvHD. Our overall hypothesis will be tested in 3 specific aims: 1. How does the schedule of RBC transfusion regulate donor T cell activation in murine allo-HSCT? 2. To test the effect of storage conditions of transfused red cells on donor T-cell activation in murine models of allo-HSCT, and to use RBC biomarkers to identify RBC units that modulate T-cell activation. 3. To prospectively monitor the effect of RBC transfusion on donor T-cell activation in allo-HSCT patients. According to our overall hypothesis, RBC transfusions are potent modulators of immune responses after allo- HSCT, and changes in transfusion practices can lead to improved survival after allogeneic transplants. Completing the proposed work will yield mechanistic insights into RBC transfusions as a novel pathway of immune regulation with translational potential. Knowledge gained from this project can improve outcomes for allogeneic HSCT recipients with broad impact in cancer immunotherapy. This project interacts with Projects 1, 2 and 4 and utilizes cores A, B and C.